High-Resolution GC Analyses of Fatty Acid Methyl Esters (FAMEs)

Fatty acid methyl esters (FAMEs) analysis is an important tool both for characterizing fats and oils and for determining the total fat content in foods. Fats can be extracted from a matrix using a nonpolar solvent and saponified to produce salts of the free fatty acids. After derivatizing the free acids to form methyl esters, the mixture can readily be analyzed by gas chromatography (GC) due to the volatility and thermal stability of the FAMEs. Gas chromatography has become an important technique in fats and oils analysis because accurate results can be obtained for complex as well as simple sample matrices.

FAMEs analyses were among the first applications for gas chromatography, so many of the GC methods originally written for analysis of fats and oils described packed column technology. However, capillary columns offer significant advantages, including more efficient separations. When analyzing fats and oils with complex fatty acid profiles such as the cis and trans forms of polyunsaturated fatty acids, higher efficiencies are needed to resolve the individual components. Capillary columns with Carbowax-type (polyethylene glycol) stationary phases are typically used for analyses of saturated and unsaturated fatty acid methyl esters (Figures 1 and 2), and biscyanopropyl phases are used to resolve cis and trans isomers of polyunsaturated components (Figure 3).

Creating FAMEs

Lipids are normally extracted from matrices using a nonpolar solvent such as ether and saponified to produce the free fatty acid salts. The fatty acid salts then are derivatized to form the fatty acid methyl esters, to increase volatility, improve peak symmetry, and decrease sample activity, thus providing more accurate analytical data. The official methods of the Association of Official Agriculture Chemists (AOAC International) [1] and the American Oil Chemists Society (AOCS) [2] contain procedures for the derivatization reaction, as does the European Pharmacopoeia [3]. In general, the glycerides are saponified by refluxing with methanolic sodium hydroxide. The esterification is effected with a reagent such as boron trifluoride in methanol, and the FAMEs are extracted with a nonpolar solvent (e.g., heptane) for analysis by GC.

Several groups of researchers have proposed simplified procedures for creating the methyl esters. For example, lipids can be transmethylated in situ. This option combines all of the conventional steps, except the drying and post-reaction workup, into one step. [4] For some samples, trimethylsulfonium hydroxide (TMSH), sodium methoxide, or methanolic hydrochloric acid, which are alternative derivatization reagents, can be used for transesterification. A major advantage of this approach is that the derivatization can be performed in a single, fast reaction step. [5,6,7]

	Peaks
1.	C14:0
2.	C14:1
3.	C16:0
4.	C16:1
5.	C18:0
6.	C18:1 (oleate)
7.	C18:1 (vaccenate)
8.	C18:2 n6 c
9.	C18:3 n3
10.	C20:0

	Peaks
11.	C20:1 n9
12.	C20:2 n6
13.	C20:4 n6
14.	C20:3 n3
15.	C20:5 n3
16.	C22:0
17.	C22:1 n9
18.	C24:0
19.	C22:6 n3
20.	C24:1

Column	FAMEWAX, 30 m, 0.32 mm ID, 0.25 µm (cat.# 12498)
Standard/Sample	Marine oil FAME mix (cat.# 35066)
Conc.:	10 mg/mL total
Injection
Inj. Vol.:	0.5 µL split (split ratio 150:1)
Inj. Temp.:	250 °C
Oven
Oven Temp.:	195 °C to 240 °C at 5 °C/min (hold 1 min)
Carrier Gas	H2, constant flow
Linear Velocity:	62 cm/sec

Detector	FID @ 250 °C

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	Peaks
1.	C14:0
2.	C15:0
3.	C16:0
4.	C16:1
5.	C16:2
6.	C17:0
7.	C17:1
8.	C16:4
9.	C18:0
10.	C18:1 (oleate)
11.	C18:1 (vaccenate)
12.	C18:2 n6 cis
13.	C18:3 n3
14.	C18:4 n6

	Peaks
15.	C18:4 n3
16.	C20:0
17.	C20:1 n7
18.	C20:1 n9
19.	C20:4 n6
20.	C20:4 n3
21.	C20:5 n3
22.	C22:1 n7
23.	C21:5 n3
24.	C23:0 (IS)
25.	C22:5 n6
26.	C22:5 n3
27.	C22:6 n3
28.	C24:1

Column	FAMEWAX, 30 m, 0.32 mm ID, 0.25 µm (cat.# 12498)
Standard/Sample	Ocean nutrition sample
Conc.:	12 mg/mL total FAMEs
Injection
Inj. Vol.:	0.5 µL split (split ratio 150:1)
Liner:	3 mm split w/ wool
Inj. Temp.:	250 °C
Oven
Oven Temp.:	195 °C to 240 °C at 5 °C/min (hold 1 min)
Carrier Gas	H2, constant flow
Linear Velocity:	62 cm/sec

Detector	FID @ 250 °C

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	Peaks	tR (min)	Conc. (µg/mL)	Structural Nomenclature
1.	Methyl butyrate	13.16	40	C4:0
2.	Methyl caproate	15.53	40	C6:0
3.	Methyl octanoate	19.43	40	C8:0
4.	Methyl decanoate	24.50	40	C10:0
5.	Methyl undecanoate	27.20	20	C11:0
6.	Methyl dodecanoate	29.87	40	C12:0
7.	Methyl tridecanoate	32.47	20	C13:0
8.	Methyl myristate	34.97	40	C14:0
9.	Methyl myristoleate	37.01	20	C14:1 (c9)
10.	Methyl pentadecanoate	37.37	20	C15:0
11.	Methyl pentadecenoate	39.36	20	C15:1 (c10)
12.	Methyl palmitate	39.68	60	C16:0
13.	Methyl palmitoleate	41.30	20	C16:1 (c9)
14.	Methyl heptadecanoate	41.86	20	C17:0
15.	Methyl heptadecenoate	43.45	20	C17:1 (c10)
16.	Methyl stearate	43.97	40	C18:0
17.	Methyl octadecenoate	44.92	20	C18:1 (t9)
18.	Methyl oleate	45.34	40	C18:1 (c9)

	Peaks	tR (min)	Conc. (µg/mL)	Structural Nomenclature
19.	Methyl linolelaidate	46.39	20	C18:2 (t9,t12)
20.	Methyl linoleate	47.32	20	C18:2 (c9,c12)
21.	Methyl arachidate	47.92	40	C20:0
22.	Methyl linolenate	48.77	20	C18:3 (c6,c9,c12)
23.	Methyl eicosenoate	49.20	20	C20:1 (c11)
24.	Methyl linolenate	49.54	20	C18:3 (c9,c12,c15)
25.	Methyl heneicosanoate	49.78	20	C21:0
26.	Methyl eicosadienoate	51.09	20	C20:2 (c11,c14)
27.	Methyl behenate	51.60	40	C22:0
28.	Methyl eicosatrienoate	52.52	20	C20:3 (c8,c11,c14)
29.	Methyl erucate	52.88	20	C22:1 (c13)
30.	Methyl eicosatrienoate	53.28	20	C20:3 (c11,c14,c17)
31.	Methyl arachidonate	53.42	20	C20:4 (c5,c8,c11,c14)
32.	Methyl tricosanoate	53.65	20	C23:0
33.	Methyl docosadienoate	54.85	20	C22:2 (c13,c16)
34.	Methyl lignocerate	55.32	40	C24:0
35.	Methyl eicosapentaenoate	56.09	20	C20:5 (c5,c8,c11,c14,c17)
36.	Methyl nervonate	56.74	20	C24:1 (C15)
37.	Methyl docosahexaenoate	62.17	20	C22:6 (c4,c7,c10,c13,c16,c19)

Column	Rt-2560, 100 m, 0.25 mm ID, 0.20 µm (cat.# 13198)
Standard/Sample	Food industry FAME mix (cat.# 35077)
Diluent:	Hexane/dichloromethane
Conc.:	1,000 µg/mL
Injection
Inj. Vol.:	1 µL split (split ratio 20:1)
Liner:	Premium 4 mm Precision liner w/wool (cat.# 23305.1)
Inj. Temp.:	225 °C
Oven
Oven Temp.:	100 °C (hold 4 min) to 240 °C at 3 °C/min (hold 15 min)
Carrier Gas	He, constant flow
Flow Rate:	1.0 mL/min

Detector	FID @ 285 °C
Make-up Gas Flow Rate:	45 mL/min
Make-up Gas Type:	N2
Hydrogen flow:	30 mL/min
Air flow:	300 mL/min
Data Rate:	20 Hz
Instrument	Agilent 7890A GC

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Analyzing Polyunsaturated FAMEs

The FAMEWAX polyethylene glycol stationary phase is specially tested with a polyunsaturated FAMEs mix to ensure resolution of the omega-3 and omega-6 fatty acids of interest. FAMEs such as methyl eicosapentenoate (C20:5) and methyl docosahexaenoate (C22:6), found in nutraceutical ingredients and products such as marine oils, also are resolved. FAMEWAX columns offer excellent resolution of polyunsaturated FAMEs with significantly reduced analysis times compared to traditional Carbowax stationary phases. In fact, analysis times of less than 10 minutes are possible! Figures 1 and 2 show analyses of a marine oil FAME standard and ocean nutrition sample, respectively. Both analyses are characterized by fast, effective resolution and sharp, symmetric peaks.

Like FAMEWAX columns, Stabilwax columns and Rtx-Wax columns provide excellent resolution of FAMEs derived from either plant or animal sources. When polyunsaturated FAMEs are analyzed on one of these Carbowax-type capillary columns, analysis times of 35-50 minutes are generally required to fully resolve the C21:5 FAME from the C23:0 internal standard, and the C24:0 FAME from C22:6.

Resolving cis and trans Isomers

Individual cis and trans isomers are resolved on a 100-meter Rt-2560 column, making this the column of choice for analyzing partially hydrogenated fats. The highly polar biscyanopropyl phase gives the selectivity needed for resolving FAME isomers, such as the cis and trans forms of C18:1. The trans isomers elute before the cis isomers on this phase, opposite of the elution order on Carbowax-based phases such as FAMEWAX or Rtx-Wax. Figure 3 shows the chromatographic separation of 37 FAMEs typically encountered in vegetable, animal, or marine fats and oils using an Rt-2560 column.

AOAC method 996.06 [1] describes the determination of total fat content based on the fatty acid content after conversion to methyl esters. This is the specified method for determining total fat content for nutritional labeling purposes. After quantifying the total FAMEs present in the derivatized sample, the amount of fat (as triglycerides) in the sample is calculated based on initial sample weight. The 100-meter Rt-2560 column meets the requirements of this procedure (Figure 4). This column also allows quantification of the total trans content.

	Peaks	tR (min)	Conc. (µg/mL)	Structural Nomenclature
1.	Methyl caproate	7.52	40	C6:0
2.	Methyl octanoate	7.88	40	C8:0
3.	Methyl decanoate	8.48	40	C10:0
4.	Methyl undecanoate	8.93	20	C11:0
5.	Methyl dodecanoate	9.51	40	C12:0
6.	Methyl tridecanoate	10.27	20	C13:0
7.	Methyl myristate	11.27	40	C14:0
8.	Methyl myristoleate	12.48	20	C14:1 (c9)
9.	Methyl pentadecanoate	12.57	20	C15:0
10.	Methyl pentadecenoate	14.15	20	C15:1 (C10)
11.	Methyl palmitate	14.28	60	C16:0
12.	Methyl palmitoleate	15.98	20	C16:1 (c9)
13.	Methyl heptadecanoate	16.51	20	C17:0
14.	Methyl heptadecenoate	18.68	20	C17:1 (c10)
15.	Methyl stearate	19.43	40	C18:0
16.	Methyl octadecenoate	21.08	20	C18:1 (t9)
17.	Methyl oleate	21.85	40	C18:1 (c9)
18.	Methyl linolelaidate	24.09	20	C18:2 (t9,t12)

	Peaks	tR (min)	Conc. (µg/mL)	Structural Nomenclature
19.	Methyl linoleate	26.14	20	C18:2 (c9,c12)
20.	Methyl arachidate	28.25	40	C20:0
21.	Methyl linolenate	29.98	20	C18:3 (c6,c9,c12)
22.	Methyl eicosenoate	32.05	20	C20:1 (c11)
23.	Methyl linolenate	32.41	20	C18:3 (c9,c12,c15)
24.	Methyl heneicosanoate	33.66	20	C21:0
25.	Methyl eicosadienoate	35.33	20	C20:2 (c11,c14)
26.	Methyl behenate	36.64	40	C22:0
27.	Methyl eicosatrienoate	37.44	20	C20:3 (c8,c11,c14)
28.	Methyl erucate	38.51	20	C22:1 (c13)
29.	Methyl eicosatrienoate	38.72	20	C20:3 (c11,c14,c17)
30.	Methyl arachidonate	39.12	20	C20:4 (c5,c8,c11,c14)
31.	Methyl tricosanoate	39.74	20	C23:0
32.	Methyl docosadienoate	41.64	20	C22:2 (c13,c16)
33.	Methyl eicosapentaenoate	43.07	20	C20:5 (c5,c8,c11,c14,c17)
34.	Methyl lignocerate	43.11	40	C24:0
35.	Methyl nervonate	45.33	20	C24:1 (c15)
36.	Methyl docosahexaenoate	54.02	20	C22:6 (c4,c7,c10,c13,c16,c19)

Column	Rt-2560, 100 m, 0.25 mm ID, 0.20 µm (cat.# 13198)
Standard/Sample	Food industry FAME mix (cat.# 35077)
Diluent:	Hexane/dichloromethane
Conc.:	1,000 µg/mL
Injection
Inj. Vol.:	1 µL split (split ratio 20:1)
Liner:	Premium 4 mm Precision liner w/wool (cat.# 23305.1)
Inj. Temp.:	235 °C
Oven
Oven Temp.:	180 °C (hold 32 min) to 215 °C at 20 °C/min (hold 31.25 min)
Carrier Gas	He, constant flow
Flow Rate:	2.0 mL/min

Detector	FID @ 325 °C
Make-up Gas Flow Rate:	45 mL/min
Make-up Gas Type:	N2
Hydrogen flow:	30 mL/min
Air flow:	300 mL/min
Data Rate:	20 Hz
Instrument	Agilent 7890A GC
Notes	C4:0 Methyl butyrate (623-42-7) elutes in the solvent front.

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To calibrate the GC system for assays of this type, use a FAME mixture such as our 37-component food industry FAME mix (Figures 3 and 4), or our 28-component food industry FAME mix. These standards include a gravimetric certificate of analysis to help ensure accurate quantification. To ensure correct identifications of the individual cis and trans isomers, use our cis/trans FAMEs mix as shown in Figure 5, or our trans fat mix shown in Figure 6.

Rtx-2330, a 90% biscyanopropyl phase, also resolves cis and trans FAME isomers. These columns are slightly less polar than Rt-2560 columns. Figure 7 shows the analysis of an animal-based fat using an Rtx-2330 column. As on Rt-2560 columns, the trans forms of the FAMEs elute before the cis forms.

Analyzing Botanical Products

Gas chromatography can be used to analyze fatty acid marker compounds in some botanical products. Both Rtx-Wax and Stabilwax capillary columns provide the efficiency and selectivity needed to perform analysis and allow accurate identification of the individual fatty acids in products such as saw palmetto oil, olive oil, and palm oil (Figures 8-10).

	Peaks	tR (min)	Conc. (mg/mL)	Structural Nomenclature
1.	Methyl stearate	34.402	2.0	C18:0
2.	Methyl petroselaidate	35.220	0.8	C18:1 (trans-6)
3.	Methyl elaidate	35.288	1.0	C18:1 (trans-9)
4.	Methyl trans-vaccenate	35.405	1.2	C18:1 (trans-11)
5.	Methyl petroselinate	35.555	0.8	C18:1 (cis-6)
6.	Methyl oleate	35.650	1.0	C18:1 (cis-9)
7.	Methyl vaccenate	35.838	1.2	C18:1 (cis-11)
8.	Methyl linoleate	37.567	2.0	C18:2 (cis-9,12)

Column	Rt-2560, 100 m, 0.25 mm ID, 0.20 µm (cat.# 13198)
Standard/Sample	cis/trans FAME mix (cat.# 35079)
Diluent:	Methylene chloride
Injection
Inj. Vol.:	1 µL split (split ratio 200:1)
Liner:	Topaz 4 mm ID straight inlet liner w/ wool (cat.# 23300)
Inj. Temp.:	240 °C
Oven
Oven Temp.:	100 °C (hold 4 min) to 240 °C at 3 °C/min (hold 10 min)
Carrier Gas	H2, constant flow
Flow Rate:	2.2 mL/min

Detector	FID @ 250 °C
Instrument	Agilent 7890A GC

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	Peaks	tR (min)	Conc. (wt.%)	Structural Nomenclature
1.	Methyl myristelaidate	21.806	6	C14:1 (trans-9)
2.	Methyl trans-pentandecen-10-oate	24.832	6	C15:1 (trans-10)
3.	Methyl palmitelaidate	27.605	6	C16:1 (trans-9)
4.	Methyl trans-heptadecen-10-oate	30.660	6	C17:1 (trans-10)
5.	Methyl petroselaidate	33.407	4	C18:1 (trans-6)
6.	Methyl elaidate	33.513	16	C18:1 (trans-9)

	Peaks	tR (min)	Conc. (wt.%)	Structural Nomenclature
7.	Methyl trans-vaccenate	33.679	12	C18:1 (trans-11)
8.	Methyl linoelaidate	35.583	6	C18:2 (trans-9,12)
9.	Methyl trans-nonadecen-7-oate	36.303	6	C19:1 (trans-7)
10.	Methyl trans-nonadecen-10-oate	36.455	12	C19:1 (trans-10)
11.	Methyl trans-eicosan-11-oate	39.270	6	C20:1 (trans-11)
12.	Methyl brassidate	44.764	6	C21:1 (trans-13)
13.	Methyl ricinelaidate	56.987	8	C18:1 (trans-9; OH-12)

Column	Rt-2560, 100 m, 0.25 mm ID, 0.20 µm (cat.# 13198)
Standard/Sample	trans Fat reference standard (cat.# 35629)
Diluent:	Hexane
Injection
Inj. Vol.:	1 µL split (split ratio 200:1)
Liner:	Topaz 4.0 mm ID straight inlet liner w/wool (cat.# 23300)
Inj. Temp.:	250 °C
Oven
Oven Temp.:	120 °C (hold 1 min) to 250 °C at 2 °C/min (hold 4 min)
Carrier Gas	H2, constant flow
Flow Rate:	2 mL/min

Detector	FID @ 250 °C
Instrument	Agilent 7890A GC

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	Peaks
1.	C14:0
2.	C16:0
3.	C16:1n7
4.	C18:0
5.	C18:1n9
6.	C18:1n7
7.	C18:2n6

	Peaks
8.	C18:3n6
9.	C18:3n3
10.	C20:3n6
11.	C20:4n6
12.	C20:5n3
13.	C22:4n6
14.	C22:5n3
15.	C22:6n3

Column	Rtx-2330, 30 m, 0.32 mm ID, 0.20 µm (cat.# 10724)
Standard/Sample	PUFA 2 mix
Injection
Inj. Vol.:	0.1 µL split (split ratio 20:1)
Inj. Temp.:	260 °C
Oven
Oven Temp.:	160 °C to 250 °C at 2 °C/min (hold 10 min)
Carrier Gas	H2, constant pressure
Linear Velocity:	40 cm/sec

Detector	FID @ 260 °C
Notes	FID sensitivity: 8 x 10-11 AFS

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	Peaks	Conc. (mg/mL)
1.	Methyl caproate (C6:0)	0.4
2.	Methyl caprylate (C8:0)	0.4
3.	Methyl nonanoate (C9:0)	2.0
4.	Methyl caprate (C10:0)	0.4
5.	Methyl laurate (C12:0)	5.0
6.	Methyl myristate (C14:0)	2.0

	Peaks	Conc. (mg/mL)
7.	Methyl palmitate (C16:0)	2.0
8.	Methyl palmitoleate (C16:1)	0.4
9.	Methyl stearate (C18:0)	0.4
10.	Methyl oleate (C18:1)	5.0
11.	Methyl linoleate (C18:2)	1.0
12.	Methyl linolenate (C18:3)	0.4

Column	Rtx-Wax, 30 m, 0.25 mm ID, 0.25 µm (cat.# 12423)
Standard/Sample	Saw palmetto standard
Conc.:	See peak list
Injection
Inj. Vol.:	1 µL split (split ratio 100:1)
Inj. Temp.:	250 °C
Oven
Oven Temp.:	120 °C (hold 3 min) to 220 °C at 20 °C/min (hold 12 min)
Carrier Gas	He, constant flow
Flow Rate:	1 mL/min
Linear Velocity:	34 cm/sec

Detector	FID @ 300 °C

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	Peaks	tR (min)	Conc. (wt.%)	Structural Nomenclature
1.	Methyl palmitate	15.256	11.9	C16:0
2.	Methyl palmitoleate	15.646	1.0	C16:1 (cis-9)
3.	Methyl stearate	18.244	3.2	C18:0
4.	Methyl oleate	18.631	73.2	C18:1 (cis-9)
5.	Methyl linoleate	19.223	8.9	C18:2 (cis-9,12)
6.	Methyl linolenate	20.114	0.8	C18:3 (cis-9,12,15)
7.	Methyl arachidate	20.985	0.5	C20:0
8.	Methyl eicosenoate	21.287	0.4	C20:1 (cis-11)

Column	Stabilwax, 60 m, 0.25 mm ID, 0.25 µm (cat.# 10626)
Standard/Sample	Olive oil reference standard (Sigma-Aldrich #47118)
Diluent:	Hexane
Injection
Inj. Vol.:	1 µL split (split ratio 100:1)
Liner:	Topaz 4 mm ID straight inlet liner w/ wool (cat.# 23300)
Inj. Temp.:	225 °C
Oven
Oven Temp.:	120 °C (hold 0.5 min) to 250 °C at 6 °C/min (hold 15 min)
Carrier Gas	H2, constant flow
Flow Rate:	2 mL/min

Detector	FID @ 250 °C
Instrument	Agilent 7890A GC
Sample Preparation	The sample was prepared using methanolic HCl.

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	Peaks	tR (min)	Conc. (wt.%)	Structural Nomenclature
1.	Methyl laurate	8.963	3.0	C12:0
2.	Methyl myristate	12.106	2.0	C14:0
3.	Methyl palmitate	15.344	50.2	C16:0
4.	Methyl palmitoleate	15.668	1.5	C16:1 (cis-9)
5.	Methyl stearate	18.254	4.4	C18:0
6.	Methyl oleate	18.620	34.1	C18:1 (cis-9)
7.	Methyl linoleate	19.224	4.5	C18:2 (cis9,12)
8.	Methyl linolenate	20.118	0.1	C18:3 (cis-9,12,15)
9.	Methyl arachidate	21.001	0.2	C20:0

Column	Stabilwax, 60 m, 0.25 mm ID, 0.25 µm (cat.# 10626)
Standard/Sample	Palm oil standard (Supelco #46962)
Diluent:	Hexane
Injection
Inj. Vol.:	1 µL split (split ratio 100:1)
Liner:	Topaz 4 mm ID straight inlet liner w/ wool (cat.# 23300)
Inj. Temp.:	225 °C
Oven
Oven Temp.:	120 °C (hold 0.5 min) to 250 °C at 6 °C/min (hold 15 min)
Carrier Gas	H2, constant flow
Flow Rate:	2 mL/min

Detector	FID @ 250 °C
Instrument	Agilent 7890A GC
Sample Preparation	The sample was prepared using methanolic HCl.

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Summary

Capillary GC is especially useful for determining total fat content, trans fat content, and total omega-3 polyunsaturated fatty acid content in foods. The choice of capillary column depends on the information required. For polyunsaturated FAMEs analysis, a FAMEWAX column allows fast, accurate quantification. A more polar Rt-2560 column is the column of choice when determining the total fat content, or the amount of trans fat, in an ingredient or end product.

Whatever your fatty acid analysis requirements, Restek can provide the consistent-performance analytical columns and reference mixes that will help you to accurately characterize your materials.

Composition of each mixture listed as a weight/weight % basis (minimum 50 mg/ampul)

References

1. M.M. Mossoba, J.K.G. Kramer, P. Delmonte, M.P. Yurawecz, J.I. Rader, Official methods for the determination of trans fat, AOCS Press, 2003. 
2. AOCS Committee, Official methods and recommended practices of the AOCS, 7th edition, AOCS Press, 2017. https://www.aocs.org/store/shop-aocs/shop-aocs?productId=70978091.
3. European pharmacopoeia (Ph. Eur.) 9th edition, European Pharmacopoeia, 2017. Book, online, PDF. 
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